Compounds and Compositions for Treating Fibrosis

ABSTRACT

The present invention provides compounds and methods for treating MKP-5 modulated disease. In certain embodiments, the MKP-5 modulated disease is a fibrotic disease.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of, and claims priority to,U.S. application Ser. No. 16/954,514, filed Jun. 16, 2020, now allowed,which is a 35 U.S.C. § 371 national phase application from, and claimspriority to, International Application No. PCT/US2018/066191, filed Dec.18, 2018, and published under PCT Article 21(2) in English, which claimspriority under 35 U.S.C. § 119(e) to U.S. Provisional Patent ApplicationNo. 62/607,110, filed Dec. 18, 2017, all of which applications areincorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under AR066003 awardedby National Institutes of Health. The government has certain rights inthe invention.

SEQUENCE LISTING

The ASCII text file named “047162-7106US2(01766)_Seq Listing” created onOct. 14, 2022, comprising 8.27 Kbytes, is hereby incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

The accumulation of extracellular matrix components in response totissue damage is a physiological process integral to tissue repair.Unfortunately, chronic insults that exacerbate injury precipitateoverproduction of extracellular matrix components by fibroblasts andmyofibroblasts, thus causing an excessive accumulation of fibrousconnective tissue, which culminates in a pathological state known asfibrosis. Regardless of the etiology and intricate progressivecomplexities of many chronic diseases, fibrosis represents a commonend-stage of tissue death. The destructive sequelae of tissue fibrosisresults in organ dysfunction and eventually failure. As such, fibrosisis the single most common reason for organ transplantation.

Fibrosis can affect various organs such as the heart, liver, lung,skeletal muscle, kidney, vasculature and heart. Fibrosis represents theend-stage in a number of chronic tissue diseases representing nearly 50%of all deaths worldwide: skeletal muscle tissue (dystrophic muscledisease), cardiac and vascular tissue (myocardial infarction), livertissue (non-alcoholic fatty liver disease/liver cirrhosis), lung tissue(idiopathic pulmonary fibrosis) and kidney tissue (chronic kidneydisease/renal fibrosis).

For example, in dystrophic muscle disease (such as, for example,Duchenne muscular dystrophy), the dystrophic muscle becomes increasinglyfibrotic, reducing the patient's ability to walk and jeopardizing thepatient's breathing and cardiovascular function. There is no treatmentor cure for dystrophic muscle diseases, and experimental genereplacement therapies have been unsuccessful.

In another example, idiopathic lung fibrosis (commonly referred to IDFor IPF) occurs in response to chronic lung injury, and is a chronicfibrotic parenchymal lung disease of unknown origin. Thickening andscaring of lung tissue reduces capacity of patients to breathe. IDF iscommon and more lethal than many cancers, with prevalence of 20-50cases/100,000. The disease is generally diagnosed in subjects older than55 years, and has a median survival time of 3-4 years. There are about200,000 patients in the U.S. at any time, and 30,000 of those die everyyear.

Despite the increasing recognition that tissue fibrosis is the cause ofsignificant morbidity and mortality of many diseases, there are fewavailable FDA-approved treatments for fibrosis.

Mitogen-activated protein (MAP) kinases (also known as MAPKs) areprotein kinases that are specific to the amino acids serine, threonine,and tyrosine. MAPKs are involved in directing cellular responses to adiverse array of stimuli, such as mitogens, osmotic stress, heat shockand proinflammatory cytokines. They regulate cell functions includingproliferation, gene expression, differentiation, mitosis, cell survivaland apoptosis.

On the other hand, MAP kinase phosphatases (MKPs) dephosphorylate theMAP kinases. Specifically, MKP-5 dephosphorylates p38 MAPK and JNK. TheMAP kinases have been implicated in the progression of tissue fibrosis.Based on multiple mouse models in which injurious tissue repair proceedsto a fibrotic end-stage, MKP-5 has been shown to be a critical positiveregulator of tissue fibrosis. Genetic inhibition of MKP-5 amelioratesfibrosis in the lung and skeletal muscle, and curtails the early eventsthat occur in vascular fibrosis and atherogenesis. At the mechanisticlevel, MKP-5-deficient cells are impaired in their ability to activatethe TGF-beta signaling pathway, which plays an established role inpromoting fibrosis. These combined observations implicate MKP-5 as atarget for tissue fibrosis therapy.

There is a need in the art for novel compounds and compositions that canbe used to treat and/or prevent fibrotic disease. The present disclosureaddresses this need.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of treating or preventinga MKP-5-modulated disease or disorder in a mammal in need thereof. Invarious embodiments, the method comprises administering to the mammal atherapeutically effective amount of a compound of formula (1a) or (1b):

wherein: Z is selected from the group consisting of NR, NC(═O)R, CH₂ andO and the

bond is single; or Z is N and the

bond is double; R₁ is selected from the group consisting of C₁-C₆ alkyl,optionally substituted phenyl and NRR; each occurrence of R isindependently selected from the group consisting of H, C₁-C₆ alkyl andoptionally substituted phenyl; A is selected from the group consistingof optionally substituted phenyl and optionally substituted heteroaryl.

In various embodiments, the compound is the compound of formula (2):

wherein: Z is selected from the group consisting of CH₂ and O; R₁ isselected from the group consisting of C₁-C₆ alkyl, optionallysubstituted phenyl and NRR, where each occurrence of R is independentlyselected from the group consisting of H, C₁-C₃ alkyl and substituted orunsubstituted phenyl; R₃ is selected from the group consisting of H,C₁-C₆ alkyl, and C₁-C₃ thioether; and R₄ is selected from the groupconsisting of H and COOR₅, where R₅ is selected from the groupconsisting of H and C₁-C₆ alkyl.In various embodiments, the aryl or heteroaryl is substituted with atleast one substituent selected from the group consisting of C₁-C₆ alkyl,C₁-C₆ thioalkyl, C(═O)OH, C(═O)OC₁-C₆ alkyl, cyano and halo.

In various embodiments, the compound has a Ki≤100 μM against MKP-5.

In various embodiments, the compound binds to an allosteric site ofMKP-5.

In various embodiments, the compound is selected from the groupconsisting of3,3-dimethyl-1-((9-(methylthio)-5,6-dihydrothieno[3,4-h]quinazolin-2-yl)thio)butan-2-one;1-((5,6-dihydrobenzo[h]quinazolin-2-yl)thio)-3,3-dimethylbutan-2-one;N,N-dimethyl-2-((9-(methylthio)-5,6-dihydrothieno[3,4-h]quinazolin-2-yl)thio)acetamide;N-ethyl-N-methyl-2-((9-(methylthio)-5,6-dihydrothieno[3,4-h]quinazolin-2-yl)thio)acetamide;1-((9-fluoro-5,6-dihydrobenzo[h]quinazolin-2-yl)thio)-3,3-dimethylbutan-2-one;1-((6H-isochromeno[4,3-d]pyrimidin-2-yl)thio)-3,3-dimethylbutan-2-one;1-(2,4-dichlorophenyl)-2-((4-(3,4-dimethylthieno[2,3-b]thiophen-2-yl)pyrimidin-2-yl)thio)ethan-1-one;1-((5,6-dihydrothieno[2,3-h]quinazolin-2-yl)thio)-3,3-dimethylbutan-2-one;3,3-dimethyl-1-(pyrimido[5,4-c]quinolin-2-ylthio)butan-2-one; and1-((6-acetyl-5,6-dihydropyrimido[5,4-c]quinolin-2-yl)thio)-3,3-dimethylbutan-2-one;and3,3-dimethyl-1-((9-propyl-5,6-dihydrothieno[3,4-h]quinazolin-2-yl)thio)butan-2-one.

In various embodiments, the compound is selected from the groupconsisting of YU032149, HJ830, HJ845, HJ846, HJ858, and HJ862.

In various embodiments, the compound is administered as part of apharmaceutical composition further comprising at least onepharmaceutically acceptable carrier.

In various embodiments, the MKP-5 modulated disease or disorder is afibrotic disease or disorder.

In various embodiments, the MKP-5 modulated disease or disorder isselected from the group consisting of dystrophic muscle disease, acardiac or vascular disease, idiopathic pulmonary fibrosis, and anycombinations thereof.

In various embodiments, the mammal is a human.

In various embodiments, the MKP-5 inhibitor is administered to themammal by at least one route selected from the group consisting ofnasal, inhalational, topical, oral, buccal, rectal, pleural, peritoneal,vaginal, intramuscular, subcutaneous, transdermal, epidural,intratracheal, otic, intraocular, intrathecal, and intravenous routes.

In various embodiments, the method further comprises administering tothe mammal at least one additional agent that treats or prevents theMKP-5 modulated disease or disorder in the mammal.

In various embodiments, the inhibitor and at least one additional agentare coformulated.

In another aspect, the invention provides a compound selected from thegroup consisting of:

In another aspect, the invention provides a kit for preventing ortreating a MKP-5 modulated disease or disorder in a mammal, the kitcomprising a MKP-5 inhibitor, optionally an applicator, and aninstructional material for use thereof, wherein the instructionalmaterial recites the amount of, and frequency with which, the MKP-5inhibitor is to be administered to the mammal to treat or prevent theMKP-5 modulated disease or disorder.

In yet another aspect, the invention provides a method of determining ifa test compound is a MKP-5 inhibitor. In certain embodiments, the methodcomprises contacting a test compound with (i) a substrate peptidecomprising the amino acid sequence pThr-Gly-pTyr and (ii) a catalyticpolypeptide comprising and/or consisting of the catalytic domain ofMKP-5, or an active fragment thereof, thus forming a composition. Inother embodiments, the method comprises measuring MKP-5 activity in thecomposition. In yet other embodiments, the method comprises comparingthe MKP-5 activity in the composition to a control. In yet otherembodiments, the substrate peptide comprises the amino acid sequenceAsp-Asp-Glu-Nle-pThr-Gly-pTyr-Val-Ala-Thr-Arg (pTpY, SEQ ID NO:3). Inyet other embodiments, the measuring of MKP-5 activity comprisesmeasuring any change in inorganic free phosphate in the composition.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of illustrative embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention,certain illustrative embodiments are shown in the drawings. It should beunderstood, however, that the invention is not limited to the precisearrangements and instrumentalities of the embodiments shown in thedrawings.

FIG. 1 comprises a schematic depicting illustrative roles of MKP-5 intissue fibrosis.

FIG. 2A comprises a graph depicting dose response of MKP-5 activity forexemplary compounds of the invention. FIG. 2B comprises a graphillustrating the dose response of MKP-5 for an exemplary compound of theinvention (YU032149). FIG. 2C comprises a graph depicting determinationof K_(d) of YU032149 with MKP-5.

FIG. 3 comprises a ribbon diagram based on the crystal structure ofMKP-5 with ligand and inhibitor bound.

FIG. 4A comprises an illustration of binding of YU032149 to MKP-5, withcertain residues illustrated. FIG. 4B comprises an illustration ofYU032149 to the allosteric site, with certain contacts between inhibitorand protein illustrated.

FIG. 5 comprises an illustration of binding of YU032149 to theallosteric site of MKP-5. Displacement of selected active site residuesin response to the binding is illustrated.

FIG. 6 comprises a bar graph illustrating normalized turnover forselected analogs of YU032149 (50 μM inhibitor in each test).

FIGS. 7A-7C comprise a series of bar graphs illustrating MAPK activityassays for selected compounds.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates in part to the identification of novelMKP-5 inhibitors, which find use in treating and/or preventing fibrosis.The present invention provides novel inhibitors of MKP-5, as well ascompositions comprising the same, and their use in treating and/orpreventing fibrosis. For example, compound YU032149 inhibits MKP-5 withan IC₅₀ of about 12 μM. In certain embodiments, compounds of theinvention inhibit MKP-5 through an allosteric mode of inhibition.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described. As used herein, each of the following termshas the meaning associated with it in this section.

Generally, the nomenclature used herein and the laboratory procedures incell culture, molecular genetics, pharmacology and organic chemistry arethose well-known and commonly employed in the art.

Standard techniques are used for biochemical and/or biologicalmanipulations. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences (e.g., Sambrook and Russell, 2012, Molecular Cloning, ALaboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.,and Ausubel et al., 2002, Current Protocols in Molecular Biology, JohnWiley & Sons, NY), which are provided throughout this document.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

A disease or disorder is “alleviated” if the severity or frequency of atleast one sign or symptom of the disease or disorder experienced by apatient is reduced.

As used herein, the terms “analog,” “analogue,” or “derivative” aremeant to refer to a chemical compound or molecule made from a parentcompound or molecule by one or more chemical reactions. As such, ananalog can be a structure having a structure similar to that of thesmall molecule inhibitors described herein or can be based on a scaffoldof a small molecule inhibitor described herein, but differing from it inrespect to certain components or structural makeup, which may have asimilar or opposite action metabolically.

As used herein, the term “binding” refers to the adherence of moleculesto one another, such as, but not limited to, enzymes to substrates,antibodies to antigens, DNA strands to their complementary strands.Binding occurs because the shape and chemical nature of parts of themolecule surfaces are complementary. A common metaphor is the“lock-and-key” used to describe how enzymes fit around their substrate.

As used herein, the terms “conservative variation” or “conservativesubstitution” as used herein refers to the replacement of an amino acidresidue by another, biologically similar residue. Conservativevariations or substitutions are not likely to change the shape of thepeptide chain. Examples of conservative variations, or substitutions,include the replacement of one hydrophobic residue such as isoleucine,valine, leucine or methionine for another, or the substitution of onepolar residue for another, such as the substitution of arginine forlysine, glutamic for aspartic acid, or glutamine for asparagine.Additional examples include swaps within groups such as Gly/Ala;Val/Ile/Leu; Asp/Glu; Asn/Gln; Ser/Thr; Lys/Arg; and Phe/Tyr.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate.

In contrast, a “disorder” in an animal is a state of health in which theanimal is able to maintain homeostasis, but in which the animal's stateof health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the animal's state of health.

An “effective amount” or “therapeutically effective amount” of acompound is that amount of compound sufficient to provide a beneficialeffect to the subject to which the compound is administered. An“effective amount” of a delivery vehicle is that amount sufficient toeffectively bind or deliver a compound.

The phrase “inhibit,” as used herein, means to reduce a molecule, areaction, an interaction, a gene, an mRNA, and/or a protein'sexpression, stability, function or activity by a measurable amount or toprevent entirely. Inhibitors are compounds that, e.g., bind to,partially or totally block stimulation, decrease, prevent, delayactivation, inactivate, desensitize, or down regulate a protein, a gene,and an mRNA stability, expression, function and activity, e.g.,antagonists.

The term “MKP-5” or “MKPS” or “DUSP10” as used herein refers to theprotein known as dual specificity protein phosphatase 10 ormitogen-activated protein kinase phosphatase 5 (see, for example, Tao &Tong, 2007, Protein Sci. 16(5):880-886, which is incorporated herein inits entirety by reference). This protein phosphatase is involved in theinactivation of MAP kinases, and has a specificity for theMAPK11-MAPK12-MAPK13-MAPK14 subfamily. In certain embodiments, MKP-5dephosphorylates p38. Human MKP-5 corresponds to the polypeptide ofamino acid sequence of SEQ ID NO:4. In certain embodiments, the MAPkinase binding domain of human MKP-5 corresponds to residues 139-287 ofSEQ ID NO:4, and the catalytic domain of human MKP-5 corresponds toresidues 305-482 of SEQ ID NO:4.

SEQ ID NO:4 is provided herein:

        10         20         30         40MPPSPLDDRV VVALSRPVRP QDLNLCLDSS YLGSANPGSN        50         60         70         80SHPPVIATTV VSLKAANLTY MPSSSGSARS LNCGCSSASC        90        100        110        120CTVATYDKDN QAQTQAIAAG TTTTAIGTST TCPANQMVNN       130        140        150        160NENTGSLSPS SGVGSPVSGT PKQLASIKII YPNDLAKKMT       170        180        190        200KCSKSHLPSQ GPVIIDCRPF MEYNKSHIQG AVHINCADKI       210        220        230        240SRRRLQQGKI TVLDLISCRE GKDSFKRIFS KEIIVYDENT       250        260        270        280NEPSRVMPSQ PLHIVLESLK REGKEPLVLK GGLSSFKQNH       290        300        310        320ENLCDNSLQL QECREVGGGA SAASSLLPQP IPTTPDIENA       330        340        350        360ELTPILPFLF LGNEQDAQDL DTMQRLNIGY VINVTTHLPL       370        380        390        400YHYEKGLFNY KRLPATDSNK QNLRQYFEEA FEFIEEAHQC       410        420        430        440GKGLLIHCQA GVSRSATIVI AYLMKHTRMT MTDAYKFVKG       450        460        470        480KRPIISPNLN FMGQLLEFEE DLNNGVTPRI LTPKLMGVET VV

The phrase “MKP-5 inhibitor” or “inhibitor of MKP-5” as used hereinrefers to a composition or compound that inhibits at least in part, ascompared to the control system that lacks the inhibitor, MKP-5 activity,MKP-5 expression and/or both, either directly or indirectly, using anymethod known to the skilled artisan. A MKP-5 inhibitor can be any typeof compound, including but not limited to, a nucleic acid, peptide,antibody, small molecule, antagonist, aptamer, or peptidomimetic.

As used herein, an “MKP-5 modulated disease” or “MKP-5 modulateddisorder” refers to a disease associated with pathological accumulationof excessive extracellular matrix proteins in an organ or tissue.Non-limiting examples of such diseases encompass, but are not limitedto, cystic fibrosis or idiopathic pulmonary fibrosis.

“Naturally occurring” as applied to an object refers to the fact thatthe object can be found in nature. For example, a polypeptide orpolynucleotide sequence that is present in an organism (includingviruses) that can be isolated from a source in nature and which has notbeen intentionally modified by man is a naturally-occurring sequence.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human.

As used herein, the term “pharmaceutically acceptable carrier” means apharmaceutically acceptable material, composition or carrier, such as aliquid or solid filler, stabilizer, dispersing agent, suspending agent,diluent, excipient, thickening agent, solvent or encapsulating material,involved in carrying or transporting a compound useful within theinvention within or to the patient such that it may perform its intendedfunction. Typically, such constructs are carried or transported from oneorgan, or portion of the body, to another organ, or portion of the body.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation, including the compound usefulwithin the invention, and not injurious to the patient. Some examples ofmaterials that may serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; surface active agents; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffersolutions; and other non-toxic compatible substances employed inpharmaceutical formulations. As used herein, “pharmaceuticallyacceptable carrier” also includes any and all coatings, antibacterialand antifungal agents, and absorption delaying agents, and the like thatare compatible with the activity of the compound useful within theinvention, and are physiologically acceptable to the patient.Supplementary active compounds may also be incorporated into thecompositions. The “pharmaceutically acceptable carrier” may furtherinclude a pharmaceutically acceptable salt of the compound useful withinthe invention. Other additional ingredients that may be included in thepharmaceutical compositions used in the practice of the invention areknown in the art and described, for example in Remington'sPharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton,Pa.), which is incorporated herein by reference.

As used herein, the language “pharmaceutically acceptable salt” or“therapeutically acceptable salt” refers to a salt of the administeredcompounds prepared from pharmaceutically acceptable non-toxic acids,including inorganic acids or bases, organic acids or bases, solvates,hydrates, or clathrates thereof.

The terms “pharmaceutically effective amount” and “effective amount”refer to a nontoxic but sufficient amount of an agent to provide thedesired biological result. That result can be reduction and/oralleviation of the signs, symptoms, or causes of a disease or disorder,or any other desired alteration of a biological system. An appropriateeffective amount in any individual case may be determined by one ofordinary skill in the art using routine experimentation.

As used herein, the terms “polypeptide,” “protein” and “peptide” areused interchangeably and refer to a polymer composed of amino acidresidues, related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds.Synthetic polypeptides can be synthesized, for example, using anautomated polypeptide synthesizer.

By the term “specifically binds,” as used herein, is meant a molecule,such as an antibody, which recognizes and binds to another molecule orfeature, but does not substantially recognize or bind other molecules orfeatures in a sample.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology, for the purpose of diminishing oreliminating those signs.

As used herein, the term “therapeutically effective amount” is an amountof a compound of the invention, that when administered to a patient,ameliorates a symptom of the disease or disorder. The amount of acompound of the invention that constitutes a “therapeutically effectiveamount” will vary depending on the compound, the disease state and itsseverity, the age of the patient to be treated, and the like. Thetherapeutically effective amount can be determined routinely by one ofordinary skill in the art having regard to his own knowledge and to thisdisclosure.

As used herein, “treating a disease or disorder” means reducing thefrequency with which a symptom of the disease or disorder is experiencedby a patient. Disease and disorder are used interchangeably herein.

As used herein, the term “treatment” or “treating” encompassesprophylaxis and/or therapy. Accordingly the compositions and methods ofthe present invention are not limited to therapeutic applications andcan be used in prophylaxis ones. Therefore “treating” or “treatment” ofa state, disorder or condition includes: (i) preventing or delaying theappearance of clinical symptoms of the state, disorder or conditiondeveloping in a subject that may be afflicted with or predisposed to thestate, disorder or condition but does not yet experience or displayclinical or subclinical symptoms of the state, disorder or condition,(ii) inhibiting the state, disorder or condition, i.e., arresting orreducing the development of the disease or at least one clinical orsubclinical symptom thereof, or (iii) relieving the disease, i.e.causing regression of the state, disorder or condition or at least oneof its clinical or subclinical symptoms.

As used herein, the term “wild-type” refers to the genotype andphenotype that is characteristic of most of the members of a speciesoccurring naturally and contrasting with the genotype and phenotype of amutant.

As used herein, the term “alkyl,” by itself or as part of anothersubstituent means, unless otherwise stated, a straight or branched chainhydrocarbon having the number of carbon atoms designated (i.e., C₁-C₁₀means one to ten carbon atoms) and includes straight, branched chain, orcyclic substituent groups. Examples include methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, andcyclopropylmethyl. Certain specific examples include (C₁-C₆)alkyl, suchas, but not limited to, ethyl, methyl, isopropyl, isobutyl, n-pentyl,n-hexyl and cyclopropylmethyl.

As used herein, the term “cycloalkyl,” by itself or as part of anothersubstituent means, unless otherwise stated, a cyclic chain hydrocarbonhaving the number of carbon atoms designated (i.e., C₃-C₆ means a cyclicgroup comprising a ring group consisting of three to six carbon atoms)and includes straight, branched chain or cyclic substituent groups.Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, and cyclooctyl. Certain specific examples include(C₃-C₆)cycloalkyl, such as, but not limited to, cyclopropyl, cyclobutyl,cyclopentyl and cyclohexyl.

As used herein, the term “substituted alkyl” or “substituted cycloalkyl”means alkyl or cycloalkyl, as defined above, substituted by one, two orthree substituents selected from the group consisting of halogen, —OH,alkoxy, tetrahydro-2-H-pyranyl, —NH₂, —N(CH₃)₂,(1-methyl-imidazol-2-yl), pyridin-2-yl, pyridin-3-yl, pyridin-4-yl,—C(═O)OH, trifluoromethyl, —C≡N, —C(═O)O(C₁-C₄)alkyl, —C(═O)NH₂,—C(═O)NH(C₁-C₄)alkyl, —C(═O)N((C₁-C₄)alkyl)₂, —SO₂NH₂, —C(═NH)NH₂, and—NO₂, advantageously containing one or two substituents selected fromhalogen, —OH, alkoxy, —NH₂, trifluoromethyl, —N(CH₃)₂, and —C(═O)OH,more advantageously selected from halogen, alkoxy and —OH. Examples ofsubstituted alkyls include, but are not limited to, 2,2-difluoropropyl,2-carboxycyclopentyl and 3-chloropropyl.

As used herein, the term “alkoxy” employed alone or in combination withother terms means, unless otherwise stated, an alkyl group having thedesignated number of carbon atoms, as defined above, connected to therest of the molecule via an oxygen atom, such as, for example, methoxy,ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homologs andisomers. In certain embodiments, alkoxy includes (C₁-C₃)alkoxy, such as,but not limited to, ethoxy and methoxy.

As used herein, the term “halo” or “halogen” alone or as part of anothersubstituent means, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom, advantageously, fluorine, chlorine, or bromine,more advantageously, fluorine or chlorine.

As used herein, the term “heteroalkyl” by itself or in combination withanother term means, unless otherwise stated, a stable straight orbranched chain alkyl group consisting of the stated number of carbonatoms and one or two heteroatoms selected from the group consisting ofO, N, and S, and wherein the nitrogen and sulfur atoms may be optionallyoxidized and the nitrogen heteroatom may be optionally quaternized. Theheteroatom(s) may be placed at any position of the heteroalkyl group,including between the rest of the heteroalkyl group and the fragment towhich it is attached, as well as attached to the most distal carbon atomin the heteroalkyl group. Examples include: —O—CH₂—CH₂—CH₃,—CH₂—CH₂—CH₂—OH, —CH₂—CH₂—NH—CH₃, —CH₂—S—CH₂—CH₃, and —CH₂CH₂—S(═O)—CH₃.Up to two heteroatoms may be consecutive, such as, for example,—CH₂—NH—OCH₃, or —CH₂—CH₂—S—S—CH₃.

As used herein, the term “aromatic” refers to a carbocycle orheterocycle with one or more polyunsaturated rings and having aromaticcharacter, i.e. having (4n+2) delocalized π (pi) electrons, where n isan integer.

As used herein, the term “aryl,” employed alone or in combination withother terms, means, unless otherwise stated, a carbocyclic aromaticsystem containing one or more rings (typically one, two or three rings)wherein such rings may be attached together in a pendent manner, such asa biphenyl, or may be fused, such as naphthalene. Examples includephenyl, anthracyl, and naphthyl. In certain embodiments, aryl includesphenyl and naphthyl, in particular, phenyl.

As used herein, the term “heterocycle” or “heterocyclyl” or“heterocyclic” by itself or as part of another substituent means, unlessotherwise stated, an unsubstituted or substituted, stable, mono- ormulti-cyclic heterocyclic ring system that consists of carbon atoms andat least one heteroatom selected from the group consisting of N, O, andS, and wherein the nitrogen and sulfur heteroatoms may be optionallyoxidized, and the nitrogen atom may be optionally quaternized. Theheterocyclic system may be attached, unless otherwise stated, at anyheteroatom or carbon atom that affords a stable structure. A heterocyclemay be aromatic or non-aromatic in nature. In certain embodiments, theheterocycle is a heteroaryl.

As used herein, the term “heteroaryl” or “heteroaromatic” refers to aheterocycle having aromatic character. A polycyclic heteroaryl mayinclude one or more rings that are partially saturated. Examples includetetrahydroquinoline and 2,3-dihydrobenzofuryl.

Examples of non-aromatic heterocycles include monocyclic groups such asaziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine,pyrroline, imidazoline, pyrazolidine, dioxolane, sulfolane,2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane,piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine,morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran,1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane,4,7-dihydro-1,3-dioxepin and hexamethyleneoxide.

Examples of heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl(such as, but not limited to, 2- and 4-pyrimidinyl), pyridazinyl,thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl,isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl,tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyland 1,3,4-oxadiazolyl.

Examples of polycyclic heterocycles include indolyl (such as, but notlimited to, 3-, 4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl,tetrahydroquinolyl, isoquinolyl (such as, but not limited to, 1- and5-isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl(such as, but not limited to, 2- and 5-quinoxalinyl), quinazolinyl,phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin,dihydrocoumarin, 1,5-naphthyridinyl, benzofuryl (such as, but notlimited to, 3-, 4-, 5-, 6- and 7-benzofuryl), 2,3-dihydrobenzofuryl,1,2-benzisoxazolyl, benzothienyl (such as, but not limited to, 3-, 4-,5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl (such as, butnot limited to, 2-benzothiazolyl and 5-benzothiazolyl), purinyl,benzimidazolyl, benztriazolyl, thioxanthinyl, carbazolyl, carbolinyl,acridinyl, pyrrolizidinyl, and quinolizidinyl.

The aforementioned listing of heterocyclyl and heteroaryl moieties isintended to be representative and not limiting.

As used herein, the term “substituted” means that an atom or group ofatoms has replaced hydrogen as the substituent attached to anothergroup.

For aryl and heterocyclyl groups, the term “substituted” as applied tothe rings of these groups refers to any level of substitution, namelymono-, di-, tri-, tetra-, or penta-substitution, where such substitutionis permitted. The substituents are independently selected, andsubstitution may be at any chemically accessible position. In certainembodiments, the substituents vary in number between one and four. Inother embodiments, the substituents vary in number between one andthree. In yet another embodiments, the substituents vary in numberbetween one and two. In yet another embodiments, the substituents areindependently selected from the group consisting of C₁₋₆ alkyl, —OH,C₁₋₆ alkoxy, halo, amino, acetamido and nitro. As used herein, where asubstituent is an alkyl or alkoxy group, the carbon chain may bebranched, straight or cyclic, in particular, straight.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Methods

In certain embodiments, the invention comprises treating a MKP-5modulated disease by administrating a MKP-5 inhibitor. In certainembodiments, the MKP-5 modulated disease is any disease that can betreated or prevented by inhibition of MKP-5. In other embodiments, theinvention comprises a method of treating fibrotic disease byadministering a MKP-5 inhibitor to a patient. In yet other embodiments,the MKP-5 modulated disease is dystrophic muscle disease, a cardiac orvascular disease, idiopathic pulmonary fibrosis, and any combinationsthereof

Compounds and Compositions

In one aspect, the invention provides a compound of formula (1a) or(1b):

wherein:

Z is selected from the group consisting of NR, NC(═O)R, CH₂ and O andthe

bond is single; or

Z is N and the

bond is double;

R₁ is selected from the group consisting of C₁-C₆ alkyl, optionallysubstituted phenyl and NRR;

each occurrence of R is independently selected from the group consistingof H, C₁-C₆ alkyl and optionally substituted phenyl;

A is selected from the group consisting of optionally substituted phenyland optionally substituted heteroaryl.

In certain embodiments, the aryl or heteroaryl is substituted with atleast one substituent selected from the group consisting of C₁-C₆ alkyl,C₁-C₆ thioalkyl, C(═O)OH, C(═O)OC₁-C₆ alkyl, cyano and halo.

In another aspect, the invention provides a compound of formula (2):

wherein:

Z is selected from the group consisting of CH₂ and O;

R₁ is selected from the group consisting of C₁-C₆ alkyl, optionallysubstituted phenyl and NRR;

each occurrence of R is independently selected from the group consistingof H, C₁-C₃ alkyl and substituted or unsubstituted phenyl;

R₃ is selected from the group consisting of H, C₁-C₆ alkyl, and C₁-C₃thioether; and

R₄ is selected from the group consisting of H and COOR₅, where R₅ isselected from the group consisting of H and C₁-C₆ alkyl.

In certain embodiments, the compound is selected from the groupconsisting of:

In certain embodiments, the compound is selected from the groupconsisting of:3,3-dimethyl-1-((9-(methylthio)-5,6-dihydrothieno[3,4-h]quinazolin-2-yl)thio)butan-2-one;1-((5,6-dihydrobenzo[h]quinazolin-2-yl)thio)-3,3-dimethylbutan-2-one;N,N-dimethyl-2-((9-(methylthio)-5,6-dihydrothieno[3,4-h]quinazolin-2-yl)thio)acetamide;N-ethyl-N-methyl-2-((9-(methylthio)-5,6-dihydrothieno[3,4-h]quinazolin-2-yl)thio)acetamide;1-((9-fluoro-5,6-dihydrobenzo[h]quinazolin-2-yl)thio)-3,3-dimethylbutan-2-one;1-((6H-isochromeno[4,3-d]pyrimidin-2-yl)thio)-3,3-dimethylbutan-2-one;1-(2,4-dichlorophenyl)-2-((4-(3,4-dimethylthieno[2,3-b]thiophen-2-yl)pyrimidin-2-yl)thio)ethan-1-one;1-((5,6-dihydrothieno[2,3-h]quinazolin-2-yl)thio)-3,3-dimethylbutan-2-one;3,3-dimethyl-1-(pyrimido[5,4-c]quinolin-2-ylthio)butan-2-one; and1-((6-acetyl-5,6-dihydropyrimido[5,4-c]quinolin-2-yl)thio)-3,3-dimethylbutan-2-one;and3,3-dimethyl-1-((9-propyl-5,6-dihydrothieno[3,4-h]quinazolin-2-yl)thio)butan-2-one.

In certain embodiments, the compound is

In certain embodiments, the compound is

In certain embodiments, the compound is

In certain embodiments, the compound is

In certain embodiments, the compound is

In certain embodiments, the compound is

In certain embodiments, the compound is

In certain embodiments, the compound is

In certain embodiments, the compound is

Combination Therapies

In certain embodiments, the compounds of the invention are useful in themethods of the invention in combination with at least one additionalagent useful for treating or preventing an MKP-5 modulated disease in amammal in need thereof. This additional agent can comprise compoundsidentified herein or compounds, e.g., commercially available compounds,known to treat, prevent or reduce the symptoms of the MKP-5 modulateddisease in a subject.

In certain embodiments, the at least one additional compound useful fortreating or preventing an MKP-5 modulated disease comprisesacetylcholinesterase inhibitors, such as, but not limited to ninetedanib(Methyl(3Z)-3-{[(4-{methyl[(4-methylpiperazin-1-yl)acetyl]amino}phenyl)amino](phenyl)methylidene}-2-oxo-2,3-dihydro-1H-indole-6-carboxylate)or pirfenidone (5-Methyl-1-phenylpyridin-2-one).

A synergistic effect may be calculated, for example, using suitablemethods such as, for example, the Sigmoid-Emax equation (Holford &Scheiner, 1981, Clin. Pharmacokinet. 6: 429-453), the equation of Loeweadditivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv.Enzyme Regul. 22:27-55). Each equation referred to above may be appliedto experimental data to generate a corresponding graph to aid inassessing the effects of the drug combination. The corresponding graphsassociated with the equations referred to above are theconcentration-effect curve, isobologram curve and combination indexcurve, respectively.

Method of Screening Compounds as MKP-5 Inhibitors

In one aspect, the invention provides a method of identifying a compoundthat inhibits MKP-5. In certain embodiments, the method comprisescontacting a putative inhibitor compound with (i) a substrate peptidecomprising the sequence pThr-Gly-pTyr and (ii) a catalytic polypeptidecomprising and/or consisting of the catalytic domain of MKP-5 (such asbut not limited to residues 305-482 of SEQ ID NO:4), or an activefragment thereof, thus forming a composition. In other embodiments, themethod comprises measuring MKP-5 activity in the composition. In yetother embodiments, the method comprises comparing the MKP-5 activity inthe composition to a control; thereby identifying the putative inhibitorcompound as a compound that inhibits MKP-5.

In certain embodiments, the catalytic polypeptide has at least about60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, and/or 100% homology with human MKP-5 inthe amino acid sequence corresponding to the catalytic domain of MKP-5.Conservative substitutions is contemplated within such homology.

In certain embodiments, the catalytic polypeptide has at least about60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, and/or 100% sequence identity with humanMKP-5 in the amino acid sequence corresponding to the catalytic domainof MKP-5.

In certain embodiments, the method is practiced as a high-throughputscreen by which a plurality of compounds (putative inhibitors) arecontacted with a peptide comprising pThr-Gly-pTyr and a polypeptidecomprising and/or consisting of the catalytic domain of MKP-5 or anactive fragment thereof, and inhibitors are identified from among theplurality of compounds by comparing their individual activities to acontrol.

Without wishing to be limited by theory, including a peptideencompassing the pThr180-Gly-pTyr182 motif of p38a MAPK (which ispresent on the activation loop of p38a MAPK and represents the primaryMKP-5 substrate) in the assay uncovers more selective and thus moreuseful inhibitor compounds. In various embodiments, the peptidecomprises SEQ ID NO: 3 (Asp-Asp-Glu-Nle-pThr-Gly-pTyr-Val-Ala-Thr-Arg),wherein Nle is norleucine.

A person of skill in the art will recognize that activity can bemeasured by combining the substrate and a polypeptide comprising and/orconsisting of MKP-5 or any catalytically active fragment therefore, i.e.a polypeptide comprising and/or consisting of the MKP-5 catalytic domainor an active fragment thereof. A person of skill in the art willappreciate that a variety of methods of measuring MKP-5 activity andcontrols are possible and will be familiar with the same by analogy toactivity assays. The control can be, by way of non-limiting example, apredetermined reference or may be a reaction performed without a MKP-5inhibitor and/or without a peptide comprising pThr-Gly-pTyr.

In a non-limiting example, the assay comprises contacting a peptidecomprising the sequence pThr-Gly-pTyr, the polypeptide comprising and/orconsisting of the catalytic domain of MKP-5 (such as but not limited toresidues 305-482 of SEQ ID NO:4), or an active fragment thereof, and thetest compound. If the test compound is a MKP-5 inhibitor, it willinhibit the dephosphorylation of the peptide comprising the sequencepThr-Gly-pTyr. If the test compound is not a MKP-5 inhibitor, it willnot inhibit the dephosphorylation of the peptide comprising the sequencepThr-Gly-pTyr. Dephosphorylation of that peptide can be evaluatedqualitatively and/or quantitatively using any methods known in the art,such as but not limited to a Malachite Green Phosphate Assay. This assayprovides a fast, reproducible, colorimetric method for measuringinorganic free phosphate in aqueous solutions. The assay method is basedon the formation of a complex between malachite green molybdate and freeorthophosphate that absorbs at 620-640 nm. This assay is a reliable andsuitable means of detecting and quantifying minimal amounts of inorganicfree phosphate and is amenable to high-throughput screeningapplications.

Kits

The invention includes a kit comprising at least one MKP-5 inhibitor,optionally an applicator, and instructional material for use thereof.

The instructional material included in the kit comprises instructionsfor preventing or treating a MKP-5-modulated disease in a mammal. Theinstructional material recites the amount of, and frequency with which,the MKP-5 inhibitor should be administered to the mammal. In certainembodiments, the kit further comprises at least one additional agentthat prevents or treats an MKP-5-modulated disease in a mammal. In otherembodiments, the kit further comprises at least one additional agentthat improves and/or prevent further loss of cognition in a mammal.

Administration/Dosage/Formulations

The regimen of administration may affect what constitutes an effectiveamount. The therapeutic formulations may be administered to the subjecteither prior to or after the onset of a disease or disorder contemplatedin the invention. Further, several divided dosages, as well as staggereddosages may be administered daily or sequentially, or the dose may becontinuously infused, or may be a bolus injection. Further, the dosagesof the therapeutic formulations may be proportionally increased ordecreased as indicated by the exigencies of the therapeutic orprophylactic situation.

Administration of the compositions of the present invention to apatient, preferably a mammal, more preferably a human, may be carriedout using known procedures, at dosages and for periods of time effectiveto treat a disease or disorder contemplated in the invention. Aneffective amount of the therapeutic compound necessary to achieve atherapeutic effect may vary according to factors such as the state ofthe disease or disorder in the patient; the age, sex, and weight of thepatient; and the ability of the therapeutic compound to treat a diseaseor disorder contemplated in the invention. Dosage regimens may beadjusted to provide the optimum therapeutic response. For example,several divided doses may be administered daily or the dose may beproportionally reduced as indicated by the exigencies of the therapeuticsituation. A non-limiting example of an effective dose range for atherapeutic compound of the invention is from about 1 and 5,000 mg/kg ofbody weight/per day. The pharmaceutical compositions useful forpracticing the invention may be administered to deliver a dose of fromng/kg/day and 100 mg/kg/day. In certain embodiments, the inventionenvisions administration of a dose which results in a concentration ofthe compound of the present invention from 1 μM and 10 μM in a mammal.One of ordinary skill in the art would be able to study the relevantfactors and make the determination regarding the effective amount of thetherapeutic compound without undue experimentation.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

In particular, the selected dosage level depends upon a variety offactors including the activity of the particular compound employed, thetime of administration, the rate of excretion of the compound, theduration of the treatment, other drugs, compounds or materials used incombination with the compound, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well, known in the medical arts.

A medical doctor, e.g., physician or veterinarian, having ordinary skillin the art may readily determine and prescribe the effective amount ofthe pharmaceutical composition required. For example, the physician orveterinarian could start doses of the compounds of the inventionemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulatethe compound in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the patients tobe treated; each unit containing a predetermined quantity of therapeuticcompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical vehicle. The dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding/formulating such a therapeutic compound for thetreatment of a disease or disorder contemplated in the invention.

In certain embodiments, the compositions of the invention are formulatedusing one or more pharmaceutically acceptable excipients or carriers. Inother embodiments, the pharmaceutical compositions of the inventioncomprise a therapeutically effective amount of a compound of theinvention and a pharmaceutically acceptable carrier.

The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms may be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it is preferable to include isotonic agents, for example, sugars,sodium chloride, or polyalcohols such as mannitol and sorbitol, in thecomposition. Prolonged absorption of the injectable compositions may bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate or gelatin.

In certain embodiments, the compositions of the invention areadministered to the patient in dosages that range from one to five timesper day or more. In other embodiments, the compositions of the inventionare administered to the patient in range of dosages that include, butare not limited to, once every day, every two, days, every three days toonce a week, and once every two weeks. It is readily apparent to oneskilled in the art that the frequency of administration of the variouscombination compositions of the invention varies from individual toindividual depending on many factors including, but not limited to, age,disease or disorder to be treated, gender, overall health, and otherfactors. Thus, the invention should not be construed to be limited toany particular dosage regime and the precise dosage and composition tobe administered to any patient is determined by the attending physicaltaking all other factors about the patient into account.

Compounds of the invention for administration may be in the range offrom about 1 μg to about 10,000 mg, about 20 μg to about 9,500 mg, about40 μg to about 9,000 mg, about 75 μg to about 8,500 mg, about 150 μg toabout 7,500 mg, about 200 μg to about 7,000 mg, about 3050 μg to about6,000 mg, about 500 μg to about 5,000 mg, about 750 μg to about 4,000mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg toabout 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80mg to about 500 mg, and any and all whole or partial incrementstherebetween.

In some embodiments, the dose of a compound of the invention is fromabout 1 mg and about 2,500 mg. In some embodiments, a dose of a compoundof the invention used in compositions described herein is less thanabout 10,000 mg, or less than about 8,000 mg, or less than about 6,000mg, or less than about 5,000 mg, or less than about 3,000 mg, or lessthan about 2,000 mg, or less than about 1,000 mg, or less than about 500mg, or less than about 200 mg, or less than about 50 mg. Similarly, insome embodiments, a dose of a second compound as described herein isless than about 1,000 mg, or less than about 800 mg, or less than about600 mg, or less than about 500 mg, or less than about 400 mg, or lessthan about 300 mg, or less than about 200 mg, or less than about 100 mg,or less than about 50 mg, or less than about 40 mg, or less than about30 mg, or less than about 25 mg, or less than about 20 mg, or less thanabout 15 mg, or less than about 10 mg, or less than about 5 mg, or lessthan about 2 mg, or less than about 1 mg, or less than about 0.5 mg, andany and all whole or partial increments thereof.

In certain embodiments, the present invention is directed to a packagedpharmaceutical composition comprising a container holding atherapeutically effective amount of a compound of the invention, aloneor in combination with a second pharmaceutical agent; and instructionsfor using the compound to treat, prevent, or reduce one or more symptomsof a disease or disorder contemplated in the invention.

Formulations may be employed in admixtures with conventional excipients,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for oral, parenteral, nasal, intravenous,subcutaneous, enteral, or any other suitable mode of administration,known to the art. The pharmaceutical preparations may be sterilized andif desired mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure buffers, coloring, flavoring and/or aromatic substances and thelike. They may also be combined where desired with other active agents,e.g., anti-fibrotic agents.

Routes of administration of any of the compositions of the inventioninclude oral, nasal, rectal, intravaginal, parenteral, buccal,sublingual or topical. The compounds for use in the invention may beformulated for administration by any suitable route, such as for oral orparenteral, for example, transdermal, transmucosal (e.g., sublingual,lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- andperivaginally), (intra)nasal and (trans)rectal), intravesical,intrapulmonary, intraduodenal, intragastrical, intrathecal,subcutaneous, intramuscular, intradermal, intra-arterial, intravenous,intrabronchial, inhalation, and topical administration.

Suitable compositions and dosage forms include, for example, tablets,capsules, caplets, pills, gel caps, troches, dispersions, suspensions,solutions, syrups, granules, beads, transdermal patches, gels, powders,pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs,suppositories, liquid sprays for nasal or oral administration, drypowder or aerosolized formulations for inhalation, compositions andformulations for intravesical administration and the like. It should beunderstood that the formulations and compositions that would be usefulin the present invention are not limited to the particular formulationsand compositions that are described herein.

Oral Administration

For oral application, particularly suitable are tablets, dragees,liquids, drops, suppositories, or capsules, caplets and gelcaps. Thecompositions intended for oral use may be prepared according to anymethod known in the art and such compositions may contain one or moreagents selected from the group consisting of inert, non-toxicpharmaceutically excipients that are suitable for the manufacture oftablets. Such excipients include, for example an inert diluent such aslactose; granulating and disintegrating agents such as cornstarch;binding agents such as starch; and lubricating agents such as magnesiumstearate. The tablets may be uncoated or they may be coated by knowntechniques for elegance or to delay the release of the activeingredients. Formulations for oral use may also be presented as hardgelatin capsules wherein the active ingredient is mixed with an inertdiluent.

In certain embodiments, the tablets of the invention comprisesaracatinib difumarate, mannitol, dibasic calcium phosphate anhydrous,crospovidone, hypromellose and magnesium stearate, with a film-coatcontaining hypromellose, macrogol 400, red iron oxide, black iron oxideand titanium dioxide. In other embodiments, the tablets of the inventioncomprise about 50 or 125 mg of saracatinib expressed as free base. Inyet other embodiments, the tablets of the invention comprise about 71.4or 178.6 mg of saracatinib expressed as difumarate salt.

For oral administration, the compounds of the invention may be in theform of tablets or capsules prepared by conventional means withpharmaceutically acceptable excipients such as binding agents (e.g.,polyvinylpyrrolidone, hydroxypropylcellulose orhydroxypropylmethylcellulose); fillers (e.g., cornstarch, lactose,microcrystalline cellulose or calcium phosphate); lubricants (e.g.,magnesium stearate, talc, or silica); disintegrates (e.g., sodium starchglycollate); or wetting agents (e.g., sodium lauryl sulfate). Ifdesired, the tablets may be coated using suitable methods and coatingmaterials such as OPADRY™ film coating systems available from Colorcon,West Point, Pa. (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-PType, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY™ White,32K18400). Liquid preparation for oral administration may be in the formof solutions, syrups or suspensions. The liquid preparations may beprepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (e.g., sorbitol syrup, methylcellulose or hydrogenated edible fats); emulsifying agent (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily estersor ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid).

Granulating techniques are well known in the pharmaceutical art formodifying starting powders or other particulate materials of an activeingredient. The powders are typically mixed with a binder material intolarger permanent free-flowing agglomerates or granules referred to as a“granulation”. For example, solvent-using “wet” granulation processesare generally characterized in that the powders are combined with abinder material and moistened with water or an organic solvent underconditions resulting in the formation of a wet granulated mass fromwhich the solvent must then be evaporated.

Melt granulation generally consists in the use of materials that aresolid or semi-solid at room temperature (i.e. having a relatively lowsoftening or melting point range) to promote granulation of powdered orother materials, essentially in the absence of added water or otherliquid solvents. The low melting solids, when heated to a temperature inthe melting point range, liquefy to act as a binder or granulatingmedium. The liquefied solid spreads itself over the surface of powderedmaterials with which it is contacted, and on cooling, forms a solidgranulated mass in which the initial materials are bound together. Theresulting melt granulation may then be provided to a tablet press or beencapsulated for preparing the oral dosage form. Melt granulationimproves the dissolution rate and bioavailability of an active (i.e.drug) by forming a solid dispersion or solid solution.

U.S. Pat. No. 5,169,645 discloses directly compressible wax-containinggranules having improved flow properties. The granules are obtained whenwaxes are admixed in the melt with certain flow improving additives,followed by cooling and granulation of the admixture. In certainembodiments, only the wax itself melts in the melt combination of thewax(es) and additives(s), and in other cases both the wax(es) and theadditives(s) melt.

The present invention also includes a multi-layer tablet comprising alayer providing for the delayed release of one or more compounds of theinvention, and a further layer providing for the immediate release of amedication for treatment of a disease or disorder contemplated in theinvention. Using a wax/pH-sensitive polymer mix, a gastric insolublecomposition may be obtained in which the active ingredient is entrapped,ensuring its delayed release.

Parenteral Administration

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intravenous, intraperitoneal, intramuscular, intrasternal injection, andkidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multidose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In certain embodiments ofa formulation for parenteral administration, the active ingredient isprovided in dry (i.e., powder or granular) form for reconstitution witha suitable vehicle (e.g., sterile pyrogen free water) prior toparenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butanediol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer system. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

Additional Administration Forms

Additional dosage forms of this invention include dosage forms asdescribed in U.S. Pat. Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389;5,582,837; and 5,007,790. Additional dosage forms of this invention alsoinclude dosage forms as described in U.S. Patent Applications Nos.20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and20020051820. Additional dosage forms of this invention also includedosage forms as described in PCT Applications Nos. WO 03/35041; WO03/35040; WO 03/35029; WO 03/35177; WO 03/35039; WO 02/96404; WO02/32416; WO 01/97783; WO 01/56544; WO 01/32217; WO 98/55107; WO98/11879; WO 97/47285; WO 93/18755; and WO 90/11757.

Controlled Release Formulations and Drug Delivery Systems

In certain embodiments, the formulations of the present invention maybe, but are not limited to, short-term, rapid-offset, as well ascontrolled, for example, sustained release, delayed release andpulsatile release formulations.

The term sustained release is used in its conventional sense to refer toa drug formulation that provides for gradual release of a drug over anextended period of time, and that may, although not necessarily, resultin substantially constant blood levels of a drug over an extended timeperiod. The period of time may be as long as a month or more and shouldbe a release which is longer that the same amount of agent administeredin bolus form.

For sustained release, the compounds may be formulated with a suitablepolymer or hydrophobic material that provides sustained releaseproperties to the compounds. As such, the compounds for use the methodof the invention may be administered in the form of microparticles, forexample, by injection or in the form of wafers or discs by implantation.

In certain embodiments, the compounds of the invention are administeredto a patient, alone or in combination with another pharmaceutical agent,using a sustained release formulation.

The term delayed release is used herein in its conventional sense torefer to a drug formulation that provides for an initial release of thedrug after some delay following drug administration and that may,although not necessarily, includes a delay of from about 10 minutes upto about 12 hours.

The term pulsatile release is used herein in its conventional sense torefer to a drug formulation that provides release of the drug in such away as to produce pulsed plasma profiles of the drug after drugadministration.

The term immediate release is used in its conventional sense to refer toa drug formulation that provides for release of the drug immediatelyafter drug administration.

As used herein, short-term refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes and any or all whole orpartial increments thereof after drug administration after drugadministration.

As used herein, rapid-offset refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes, and any and all whole orpartial increments thereof after drug administration.

Dosing

The therapeutically effective amount or dose of a compound of thepresent invention depends on the age, sex and weight of the patient, thecurrent medical condition of the patient and the progression of adisease or disorder contemplated in the invention. The skilled artisanis able to determine appropriate dosages depending on these and otherfactors.

A suitable dose of a compound of the present invention may be in therange of from about 0.01 mg to about 5,000 mg per day, such as fromabout 0.1 mg to about 1,000 mg, for example, from about 1 mg to about500 mg, such as about 5 mg to about 250 mg per day. The dose may beadministered in a single dosage or in multiple dosages, for example from1 to 4 or more times per day. When multiple dosages are used, the amountof each dosage may be the same or different. For example, a dose of 1 mgper day may be administered as two 0.5 mg doses, with about a 12-hourinterval between doses.

It is understood that the amount of compound dosed per day may beadministered, in non-limiting examples, every day, every other day,every 2 days, every 3 days, every 4 days, or every 5 days. For example,with every other day administration, a 5 mg per day dose may beinitiated on Monday with a first subsequent 5 mg per day doseadministered on Wednesday, a second subsequent 5 mg per day doseadministered on Friday, and so on.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the inhibitor of the invention isoptionally given continuously; alternatively, the dose of drug beingadministered is temporarily reduced or temporarily suspended for acertain length of time (i.e., a “drug holiday”). The length of the drugholiday optionally varies between 2 days and 1 year, including by way ofexample only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days,12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days,120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days,320 days, 350 days, or 365 days. The dose reduction during a drugholiday includes from 10%-100%, including, by way of example only, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%.

Once improvement of the patient's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, is reduced, as a function of thedisease or disorder, to a level at which the improved disease isretained. In certain embodiments, patients require intermittenttreatment on a long-term basis upon any recurrence of symptoms and/orinfection.

The compounds for use in the method of the invention may be formulatedin unit dosage form. The term “unit dosage form” refers to physicallydiscrete units suitable as unitary dosage for patients undergoingtreatment, with each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect,optionally in association with a suitable pharmaceutical carrier. Theunit dosage form may be for a single daily dose or one of multiple dailydoses (e.g., about 1 to 4 or more times per day). When multiple dailydoses are used, the unit dosage form may be the same or different foreach dose.

Toxicity and therapeutic efficacy of such therapeutic regimens areoptionally determined in cell cultures or experimental animals,including, but not limited to, the determination of the LD50 (the doselethal to 50% of the population) and the ED50 (the dose therapeuticallyeffective in 50% of the population). The dose ratio between the toxicand therapeutic effects is the therapeutic index, which is expressed asthe ratio between LD50 and ED50. The data obtained from cell cultureassays and animal studies are optionally used in formulating a range ofdosage for use in human. The dosage of such compounds lies preferablywithin a range of circulating concentrations that include the ED50 withminimal toxicity. The dosage optionally varies within this rangedepending upon the dosage form employed and the route of administrationutilized.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents were considered to be within the scope of thisinvention and covered by the claims appended hereto. For example, itshould be understood, that modifications in reaction conditions,including but not limited to reaction times, reaction size/volume, andexperimental reagents, such as solvents, catalysts, pressures,atmospheric conditions, and reducing/oxidizing agents, withart-recognized alternatives and using no more than routineexperimentation, are within the scope of the present application.

It is to be understood that wherever values and ranges are providedherein, all values and ranges encompassed by these values and ranges,are meant to be encompassed within the scope of the present invention.Moreover, all values that fall within these ranges, as well as the upperor lower limits of a range of values, are also contemplated by thepresent application.

The following examples further illustrate aspects of the presentinvention. However, they are in no way a limitation of the teachings ordisclosure of the present invention as set forth herein.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Example 1: Assay for MKP-5 Inhibitors

Compounds from the pilot collection consisting of Microsource GenPlus,Microsource Natural Product, NIH Clinical collections, ChemBridgeDIVERSet, ChemBridge MicroFormats, Maybridge Diversity, and ChemBridgeAmines and Alcohols were screened. Assay conditions were 1.5 μM MKP-5and 25 μM dual phosphorylated peptide DDE(Nle)(pT)G(pY)VATR (10 μL totalvolume), incubated for 30 minutes at 37° C. followed by addition of 40μL Malachite Green reagent. Z′ values are ˜0.7-0.8. Malachite greenreagent was made by combining 30 mL 0.045% Malachite Green (Sigma#M-9636) with 20 mL 4.2% ammonium molybdate in 4 M HCl, shaking for atleast 30 mins and adding 0.01% Tween 20. The dose response for the fourmost potent compounds (structures below) is shown in FIG. 2 and Table 1.

The following % inhibition data form MKP-5 were obtained at 12.5 μM ofcompound concentration: YU252252, 37%; YU241658, 15%; YU241659, 12%.

TABLE 1 MKP5 MKP5 MKP5 STEP40 MKP5 STEP40 EZReader EZREader MalachiteMalachite pNPP pNPP Kd (M) fluor_TpY fluor pT_Y pTpY pTpY pTpY pTpYYU129641 1.04E-05 1.41E-05 >.0001 >.0001 >.0002 >.0002YU119284 >.0002 >.0002 >.0001 >.0001 >.0002 >.0002 YU109017 1.03E-051.56E-05 >.0001 >.0001 >.0001 >.0002 YU032149 1.75E-05 1.13E-056.25E-06 >.0002 4.23E-06 >.0002 fluor_TpYFITC-Aha-Asp-Glu-Leu-Thr-Gly-pTyr-Val-Ala-Thr-Arg-(CONH2) fluor pT_YFITC-Aha-Asp-Glu-Leu-pThr-Gly-Tyr-Val-Ala-Thr-Arg-(CONH2) pTpYAsp-Asp-Glu-Nle-pThr-Gly-pTyr-Val-Ala-Thr-Arg-(COOH) fluor_TpY SEQ IDNO: 1 fluor pT_Y SEQ ID NO: 2 pTpY SEQ ID NO: 3

Example 2: Crystal Structure of Human MKP-5

Crystallization was achieved using the hanging drop vapor diffusionmethod with 12 mg/mL protein concentration and 5 mM inhibitor (YU032149,formula 3), incubated at 4° C. Well solution contained 200 mM ammoniumacetate, 100 mM HEPES (pH 7.5), 25% w/v PEG 3350. Crystals grew in 4-7days. Mother liquor with an additional 3% w/v PEG 3350 was used ascryoprotectant. A ribbon diagram of the structure is shown in FIG. 3 .Data statistics are shown in Table 2.

TABLE 2 MKP5CD: YU032149 Data Collection Space Group P 1 21 1 CellDimensions a, b, c (Å) 66.5, 129.4, 83.5 α, β, γ (°) 90, 91.7, 90Resolution (Å) 40.89-3.09 R_(meas) 0.244 (0.961) I/σI 7.5 (1.8)Completeness 98.2 (100) (%) Redundancy 3.8 (4.2) Refinement Resolution(Å) 41.73-3.20 (3.33-3.20) No. reflections 25,718 (unique) (2,503)R/R_(free) 20.6/25.7 (25.4/27.8) RMS deviations Bond lengths .003 (Å)Bond angles .517 (°) Ramachandran 94/5.7/0.2 Favored/Allowed/ Outlier(%) Clashscore 2.51 Average B-factor 34.00 Protein 33.99 Ligand 34.43

Without wishing to be limited by theory, YU032149 appears based on thecrystal structure to inhibit MKP-5 by binding to an allosteric sitewhich initiates a conformational change in the protein that ultimatelydecreases the volume of the catalytic site by ˜30%. Contacts betweenresidues in the allosteric site and YU032149 are shown in FIG. 4 .Displacement of active site residues is shown in FIG. 5 .

Example 3: Other Inhibitors of MKP-5

Commercially available derivatives of YU032149 were evaluated foractivity against MKP-5. The results are shown in FIG. 6 . A series ofcompounds, herein referred to as the HJ series, was derived from theYU032149 core based on the co-crystal structure. Structures are shownabove. Activity data for the HJ series is shown in FIG. 7 .

Example 4: Synthesis of the HJ Series

An illustrative scheme applicable to certain compounds contemplatedwithin the invention is provided herein:

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

What is claimed is:
 1. A method of treating, ameliorating, or preventinga MKP-5-modulated disease or disorder in a mammal in need thereof, themethod comprising administering to the mammal a therapeuticallyeffective amount of a compound of formula (1a) or (1b):

wherein: Z is selected from the group consisting of NR, NC(═O)R, CH₂ andO and the

bond is single; or Z is N and the

bond is double; R₁ is selected from the group consisting of C₁-C₆ alkyl,optionally substituted phenyl and NRR; each occurrence of R isindependently selected from the group consisting of H, C₁-C₆ alkyl andoptionally substituted phenyl; A is selected from the group consistingof optionally substituted phenyl and optionally substituted heteroaryl.2. The method of claim 1, wherein the compound is the compound offormula (2):

wherein: Z is selected from the group consisting of CH₂ and O; R₁ isselected from the group consisting of C₁-C₆ alkyl, optionallysubstituted phenyl and NRR, where each occurrence of R is independentlyselected from the group consisting of H, C₁-C₃ alkyl and substituted orunsubstituted phenyl; R₃ is selected from the group consisting of H,C₁-C₆ alkyl, and C₁-C₃ thioether; and R₄ is selected from the groupconsisting of H and COORS, where R₅ is selected from the groupconsisting of H and C₁-C₆ alkyl.
 3. The method of claim 1, wherein thearyl or heteroaryl is substituted with at least one substituent selectedfrom the group consisting of C₁-C₆ alkyl, C₁-C₆ thioalkyl, C(═O)OH,C(═O)OC₁-C₆ alkyl, cyano and halo.
 4. The method of claim 1, wherein thecompound has a Ki≤100 μM against MKP-5.
 5. The method of claim 1,wherein the compound binds to an allosteric site of MKP-5.
 6. The methodof claim 1, wherein the compound is selected from the group consistingof3,3-dimethyl-1-((9-(methylthio)-5,6-dihydrothieno[3,4-h]quinazolin-2-yl)thio)butan-2-one;1-((5,6-dihydrobenzo[h]quinazolin-2-yl)thio)-3,3-dimethylbutan-2-one;N,N-dimethyl-2-((9-(methylthio)-5,6-dihydrothieno[3,4-h]quinazolin-2-yl)thio)acetamide;N-ethyl-N-methyl-2-((9-(methylthio)-5,6-dihydrothieno[3,4-h]quinazolin-2-yl)thio)acetamide;1-((9-fluoro-5,6-dihydrobenzo[h]quinazolin-2-yl)thio)-3,3-dimethylbutan-2-one;1-((6H-isochromeno[4,3-d]pyrimidin-2-yl)thio)-3,3-dimethylbutan-2-one;1-(2,4-dichlorophenyl)-2-((4-(3,4-dimethylthieno[2,3-b]thiophen-2-yl)pyrimidin-2-yl)thio)ethan-1-one;1-((5,6-dihydrothieno[2,3-h]quinazolin-2-yl)thio)-3,3-dimethylbutan-2-one;3,3-dimethyl-1-(pyrimido[5,4-c]quinolin-2-ylthio)butan-2-one; and1-((6-acetyl-5,6-dihydropyrimido[5,4-c]quinolin-2-yl)thio)-3,3-dimethylbutan-2-one;and3,3-dimethyl-1-((9-propyl-5,6-dihydrothieno[3,4-h]quinazolin-2-yl)thio)butan-2-one.7. The method of claim 1, wherein the compound is selected from thegroup consisting of YU032149, HJ830, HJ845, HJ846, HJ858, and HJ862. 8.The method of claim 1, wherein the compound is administered as part of apharmaceutical composition further comprising at least onepharmaceutically acceptable carrier.
 9. The method of claim 1, whereinthe MKP-5 modulated disease or disorder is a fibrotic disease ordisorder.
 10. The method of claim 9, wherein the MKP-5 modulated diseaseor disorder is selected from the group consisting of dystrophic muscledisease, a cardiac or vascular disease, idiopathic pulmonary fibrosis,and any combinations thereof.
 11. The method of claim 1, wherein themammal is a human.
 12. The method of claim 1, wherein the MKP-5inhibitor is administered to the mammal by at least one route selectedfrom the group consisting of nasal, inhalational, topical, oral, buccal,rectal, pleural, peritoneal, vaginal, intramuscular, subcutaneous,transdermal, epidural, intratracheal, otic, intraocular, intrathecal,and intravenous routes.
 13. The method of claim 1, further comprisingadministering to the mammal at least one additional agent that treats orprevents the MKP-5 modulated disease or disorder in the mammal.
 14. Themethod of claim 13, wherein the inhibitor and at least one additionalagent are coformulated.
 15. A compound selected from the groupconsisting of:


16. A kit for preventing or treating a MKP-5 modulated disease ordisorder in a mammal, the kit comprising a MKP-5 inhibitor, optionallyan applicator, and an instructional material for use thereof, whereinthe instructional material recites the amount of, and frequency withwhich, the MKP-5 inhibitor is to be administered to the mammal to treator prevent the MKP-5 modulated disease or disorder.
 17. A method ofdetermining if a test compound is a MKP-5 inhibitor, the methodcomprising: contacting a test compound with (i) a substrate peptidecomprising the amino acid sequence pThr-Gly-pTyr and (ii) a catalyticpolypeptide comprising and/or consisting of the catalytic domain ofMKP-5, or an active fragment thereof, thus forming a composition;measuring MKP-5 activity in the composition; and comparing the MKP-5activity in the composition to a control; thereby determining if thetest compound is a MKP-5 inhibitor.
 18. The method of claim 17, whereinthe substrate peptide comprises the amino acid sequenceAsp-Asp-Glu-Nle-pThr-Gly-pTyr-Val-Ala-Thr-Arg (pTpY, SEQ ID NO:3). 19.The method of claim 17, wherein the measuring of MKP-5 activitycomprises measuring any change in inorganic free phosphate in thecomposition.